PROJECT SUMMARY/ABSTRACT DNA double-strand breaks (DSBs) are highly toxic DNA lesions that can occur spontaneously during DNA replication, but also are introduced upon exposure of cells to chemical mutagens or ionizing radiation. DSBs can lead to genome instability and mutations, and defects in DSB repair underlie many human diseases, including disorders associated with cancer predisposition. Nuclear Ubiquitous Casein and Cyclin-dependent Kinases Substrate 1 (NUCKS1) is a highly post- translationally modified, DNA-binding and vertebrate-specific protein. NUCKS1 is a paralog of RAD51- Associated Protein 1 (RAD51AP1) and functions in DNA repair by homologous recombination (HR). Downregulation of NUCKS1 impairs HR, renders human cells hypersensitive to chemotherapeutic agents, compromises replication fork stability, and increases the susceptibility to radiation carcinogenesis in mice. NUCKS1 is phosphorylated at several residues upon exposure of cells to DNA damaging agents. However, the phenotypic consequences of these modifications on the HR reaction are not understood. Of note, loss of NUCKS1 downregulates the formation of early DNA damage-induced RAD51 foci and leads to greatly elevated and persistent levels of DNA damage-induced RAD54 foci. Moreover, NUCKS1 functionally interacts with RAD54. Yet, how NUCKS1-RAD54 complex formation impacts upon HR and cancer avoidance is not known. This application will delineate the biology of NUCKS1 and its functional interaction with the DNA motor protein RAD54 in biochemical and cell-based genetic assays. We will also include the analysis of post-translationally modified NUCKS1 and of a few select cancer-associated NUCKS1 variants to better understand how these impact upon HR and DNA replication. Taken together, our study will fill critical knowledge gaps in our understanding of NUCKS1 function in HR and tumor suppression. Given the importance of DSB repair and HR in tumor suppression and in the removal of DNA lesions induced by ionizing radiation and other environmental mutagens, the results from our investigation likely will have direct relevance to improved risk predictions for human health from environmental factors.